Recombinant human SULT1A1 (R&D Systems) activity in the presence of 0.2 mM 3′-phosphoadenosine-5′-phosphosulfate and the indicated concentrations of RITA was measured by a phosphatase-coupled assay using the Universal Sulfotransferase Activity Kit (R&D Systems) according to the manufacturer’s instructions. The assay was carried out in Corning clear 96-well plates and incubated (37°C, 1 hour) before addition of detection reagents and visualization at 620 nm using a SpectraMax M5 microplate reader using the SoftMax Pro software (Molecular Devices).
Recombinant human BLVRB was purchased from R&D Systems, Inc. BLVRB activity was measured in the presence of 20 ng/μL BLVRB, obatoclax, and 0.01 mM NADPH in 100 mM sodium phosphate (pH 7.5) in a 20 μL volume in white, opaque 384-well plates. The reaction was incubated for 3 hours at 37°C before detection using the NADPH-Glo assay (Promega) according to the manufacturer’s instructions. NADPH and sodium phosphate were purchased from Sigma.
Recombinant human BLVRB was purchased from R&D Systems, Inc. BLVRB activity was measured in the presence of 20 ng/μL BLVRB, obatoclax, and 0.01 mM NADPH in 100 mM sodium phosphate (pH 7.5) in a 20 μL volume in white, opaque 384-well plates. The reaction was incubated for 3 hours at 37°C before detection using the NADPH-Glo assay (Promega) according to the manufacturer’s instructions. NADPH and sodium phosphate were purchased from Sigma.